Development of an HPLC-DAD method for the analysis of a pharmaceutical product composed of ten peptides

Abstract

Since their introduction for therapeutic purposes approximately a hundred years ago, peptides have played an important role in the pharmaceutical industry, and their development has been reflected in the growing production of peptide-based pharmaceutical products. Especially in the recent years, the pharmaceutical industry has experienced an exponential increase in the use of these molecules, and investment in research and development of these drugs is expected to increase in the coming years. These trends can be associated to the numerous advantages peptides present for therapeutic uses and their ease of synthesis. In this project, a high-performance liquid chromatography (HPLC) preexisting method coupled with diode array detector (DAD) for peptide analysis has been optimized. The objective has been to improve the method parameters to achieve a better separation and detection through ultraviolet absorption of ten peptides that are part of a pharmaceutical formulation. This method is essential for the quantification of the content of the compounds and to analyse potential degradation products. The optimization of the HPLC-DAD method has been based on the study and adjustment of the specific parameters that affect directly the separation, detection and quantification of the analytes. In this project, a suitable chromatographic column and mobile phase were selected to enhance the chromatographic performance. Afterwards, essential parameters such as the gradient and the column temperature were optimized to achieve a proper separation and quantification. Finally, an individual assessment of the optimal detection wavelength for every peptide was performed. The final version of the studied method shows important improvements respect the initial one. Peak splitting, peptide separation, quantification and quality parameters such as the resolution, theoretical plates and asymmetry factor are the principal aspects that have improved during the development of the method